The clinical use of leucovorin in therapeutic regimens with high doses of methotrexate (MTX) has advanced the application of antifolates to certain types of neoplasia. The proposed research presents another approach to the amelioration of drug toxicity and to the possible solution of certain problems arising during high-dose MTX therapy with leucovorin rescue, i.e. enzymic degradation of MTX. A bacterial carboxypeptidase that hydrolyzes MTX in vitro 20 times more rapidly than 5-methyltetrahydrofolate has been considered for its potential value as a rescue agent. The research plan includes a determination of the efficiency of the carboxypeptidase in vivo in hydrolyzing plasma MTX, which persists after administration of the drug, and plasma folate. A major portion of the research plan is an evaluation of the ability of the carboxypeptidase in the L1210 leukemia system, treated with high doses of MTX, to lower toxic levels of plasma MTX rapidly and effectively to subthreshold levels, thereby hastening the onset of the recovery of DNA synthesis in sensitive tissue and preventing cytotoxicity. The overall approach to the assessment of the enzyme is relevant to the pharmacokinetics of high-dose MTX and seeks to explore possibilities that may influence selective rescue of sensitive host tissue renewal systems. Another portion of the research deals with an original concept that recovery of DNA synthesis in drug-sensitive tissues, following exposure to MTX, may depend upon tissue-specific thresholds of plasma folate (5-methyltetrahydrofolate) and tissue levels of methylenetetrahydrofolate reductase. The results of other studies may support the consideration that administered leucovorin is an absolute requirement in rescue regimens following high-dose MTX therapy to replenish depressed levels of circulating plasma folate. Biochemical studies with the Lewis lung carcinoma will be initiated to procure information regarding specific aspects of folate metabolism, prerequisite to selection of this tumor for high-dose MTX therapy.